Annals of Virology and Research

Cite this article: Henriques AM, Fagulha T, Barros SC, Ramos F, Duarte M, et al. (2017) Newcastle Disease Virus 2015 Outbreak in Porto Santo Island, Por-tugal. Ann Virol Res 3(1): 1025.

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*Corresponding authorAna M. Henriques, Department of Virology, Instituto Nacional de Investigação Agrária e Veterinária, Portugal, Av. da República, Quinta do Marquês, 2780-157 Oeiras, Portugal, Tel: 351217115290; Email:

Submitted: 15 November 2016

Accepted: 17 February 2017

Published: 20 February 2017

Copyright© 2017 Henriques et al.

OPEN ACCESS

Keywords•Newcastle disease virus•Epidemiology•Histopathology•Molecular diagnosis•Phylogenetic analysis

Review Article

Newcastle Disease Virus 2015 Outbreak in Porto Santo Island, PortugalAna M. Henriques1*, Teresa Fagulha1, Sílvia C. Barros1, Fernanda Ramos1, Margarida Duarte1, Tiago Luís1, Sara Silva2, Maria Gomes3, Fátima Freitas3, Maria Gouveia3, Margarida Costa3 and Miguel Fevereiro1

1Department of Virology, Instituto Nacional de Investigação Agrária e Veterinária, Portugal2Direção Regional para a Administração Pública do Porto Santo (DRAPS), Portugal3Direção Regional de Agricultura (DRA), Portugal

Abstract

Newcastle disease (ND) is a highly contagious viral disease of birds with a devastating impact on poultry industry that leads to huge economic losses. The disease is caused by Newcastle disease virus (NDV), found worldwide in several species of birds.

In July 2015, three pigeons (Columba livia) were found moribund inside a bird park in Porto Santo Island (Madeira Archipelago, Portugal). Lung and liver congestion was observed at necropsy and histopathology, but neither macro- nor microscopic examinations of the three specimens lead to conclusions on the possible etiology of these deaths. A pool of viscera from the three pigeons (16919-15) was prepared and sent for virological examination. NDV-RNA was detected by a real-time RT-PCR targeting a fragment within the L-gene. In order to characterize the virulence of the viral strain, a 561-nucleotide long fragment of the F-gene was amplified and sequenced. The deduced amino acid sequence comprising the cleavage site (RRQKRFVGA) was characteristic of virulent viruses. Blast analysis showed higher similarity (99%) with strain HG424625, originated in a pigeon from Nigeria in 2013, followed by strains JN638234-6, obtained from doves in Italy in 2010 and 2011, with which it shared 97% similarity. The similarity with the remaining strains available to date in GenBank is lower than 91%.

After virulent NDV was confirmed in the island, an epidemiological survey was initiated, leading to the detection of the NDV in viscera of two other birds, one pigeon (17991-15) and one turtle dove (19095-15). A total of 5 out of 172 samples tested positive for NDV-RNA (2.9% prevalence). The presence of antibodies against NDV was investigated in 46 serum samples, mainly from chicken (82.6%), 14 testing positive (30.4% seroprevalence).

Phylogenetic analysis performed by Maximum Likelihood revealed that the three strains characterized in this study belong to subgenotype i from genotype VI, together with the strains from Nigeria and Italy referred above.

To the best of our knowledge, this is the first report of Newcastle disease virus in the Porto Santo Island.

INTRODUCTIONNewcastle disease (ND) is caused by avian paramyxovirus

serotype 1 (APMV-1), also known as Newcastle disease virus (NDV) that belongs to the Avulavirus genus of the Paramyxoviridae family (International Committee on Taxonomy of Viruses, 2015). The virus was first reported in poultry in the island of Java, Indonesia, in 1926 [1] and in Newcastle-on-Tyne, England in 1927 [2]. ND is spread worldwide affecting various species of birds that exhibit different levels of susceptibility; the disease is more severe in chickens and turkeys with ducks and guinea fowl being less affected [3,4].

The first detection of NDV in Portugal dates back to 1947 (Miguel Fevereiro, personal communication), referring its isolation from Leghorn chicken cadavers. Later on, NDV disseminated to the whole national territory, reaching the highest prevalence in the mid nineties (Miguel Fevereiro, personal communication), with the vast majority of the cases reported in chicken (71.6%),

although also in pigeons, partridges, pheasants, turkeys and other bird species. A gradual decrease in the number of cases was observed during the 2000s, affecting mostly pigeons. In the last five years, NDV infection has been rarely detected. In 2011, two NDV isolates were obtained from pigeons and in 2012 a vaccine virus from a broiler and a virulent virus from a racing pigeon were also isolated. In the Madeira archipelago, NDV was detected in the mid nineties. All cases were confined to the Madeira Island, with no detections reported in Porto Santo Island, located 70 Km northeast. At the Madeira Island, the virus was detected mainly in chicken backyard poultry, but also in partridges (Miguel Fevereiro, personal communication). After the last report in 1994, the virus was not detected again in the archipelago until 2015, despite only passive surveillance was performed. The 2015 outbreak in Porto Santo Island described in the present study is, to our knowledge, the first occurrence of the infection in the island.

The clinical signs observed upon infection with NDV vary greatly depending on the strain of the virus and the host species.

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The most common are respiratory signs, including gasping and coughing, but ruffled feathers, open mouth breathing, hyperthermia, anorexia, listlessness and hypothermia may also occur. Neurological signs may include depression, torticollis, muscular tremors, ataxia, drooping wings and paralysis. A drop in egg production is often observed. The incubation period ranges from two to 15 days and mortality may reach 100% within a flock [5].

Newcastle disease virus (NDV) is a single-stranded, non-segmented, negative-sense, enveloped RNA virus with a genome of about 15.2 kb that encodes six major structural proteins, including the nucleocapsid protein (NP), phosphoprotein (P), matrix protein (M), fusion protein (F), hemagglutinin-neuraminidase protein (HN), and the large polymerase protein (L) [6]. It was shown that NDV virulence is dependent on the amino acid sequence at the cleavage site encoded by the F gene. Highly virulent strains of NDV can be discriminated from low virulent strains by the presence of a multibasic amino acid motif at the proteolytic cleavage site of the fusion (F) protein [7], since this multibasic amino acid motif is cleaved by ubiquitous proteases [8]. Based on the virulence of disease developed in chickens, NDVs have been categorized into lentogenic, mesogenic, and velogenic strains [9].

An antigenic variant of the NDV known as pigeon paramyxovirus type 1 (PPMV-1) is found in pigeons. During PPMV-1 infections, central nervous system symptoms are developed and high mortality rates may be observed in pigeons [10]. On the other hand, no significant clinical signs or mortality were observed in chickens inoculated with PPMV-1 [11]. In this paper we report the clinical, genetic, serologic and epidemiological data related with the recent outbreak of Newcastle disease virus that took place on July 2015, in the Porto Santo Island, Madeira, Portugal.

METHODS

Case history and control measures

In June 2015, 11 carcasses of European turtle dove (Streptopelia turtur) were found dead in the outer vicinity of a Mini-Zoo private exotic bird park also known in Porto Santo as the mini-botanic garden due to its exotic tree species. Suspicion of poisoning was initially considered. This 5,380 m2 private park harbors 21 species in a total of 249 exotic birds and its dense vegetation, when compared to the arid natural flora of the western part of the island where the park is located, provides an attractive microhabitat for free ranging wild birds that visit the park for shelter, food and water since it is an open area.

The advanced stage of putrefaction of all these turtle doves hampered anatomopathological examination as well as other microbiological tests. Gizzard and intestine content tested negative for pesticides, eliminating the initial hypothesis of poisoning. At this stage molecular tests were not performed.

One week later, three free ranging pigeons (Columba livia) were found moribund inside the park. The birds were euthanized by the official services, and the carcasses sent to the regional laboratory in Madeira Island (Laboratório Regional de Veterinária e Segurança Alimentar da Madeira) for necropsy and

histopathology. Viscera were collected and sent to INIAV (Instituto Nacional de Investigação Agrária e Veterinária) for virological examination. Despite the fact that none of the specimens showed lesions characteristic of Newcastle disease, all tested positive for NDV-RNA. Consequently, an active surveillance plan was implemented by the local Veterinary Authorities in a protection zone based on a radius of 3 Km, itself contained in a surveillance zone based on a radius of 10 Km, all over the island. All domestic and wild birds found dead (n=19) were collected and analyzed for NDV. Cloacal swabs (n=136), fecal (n=17) and serum samples (n=46) from asymptomatic birds were also tested.

Necropsy and histopathological analysis

At necropsy, viscera were collected for histopathological and virological examination.

Organs were fixed in 4% formaldehyde (10% formalin), followed by paraffin embedding. After processing, sections of 4-5 µm were stained with hematoxilin and eosin (H&E).

Virus isolation

Virus isolation was performed in embryonated chicken eggs. The pool of organs was homogenized and suspended in phosphate-buffered saline containing 200 U/ml penicillin (Gibco, Life Technologies Corporation, Carlsbad, CA, USA), 200 µg/ml streptomycin (Gibco®) and 100 µg/ml gentamycin (Gibco). Following centrifugation at 1500 g for 10 min, 200 µl of the supernatant were inoculated on the chorioallantoic cavity of five 11-day-old specific pathogen free (SPF) embryonated eggs (Lohman Tierzucht, GmbH, Cuxhaven, Germany). The eggs were incubated at 37°C for 4-7 days. The procedure was repeated until the third blind passage. Allantoic liquid was collected and submitted to hemagglutination inhibition test (HAI) and RNA extraction as indicated below.

Hemagglutination inhibition test (HAI)

The hemagglutinating NDV was identified by means of the hemagglutination inhibition test (HAI) using a specific antiserum, performed as indicated in EU directive 92/66/EEC (CEC, 1992).

The presence of NDV antibodies in serum samples was also investigated by hemagglutination inhibition test, performed as indicated in the same EU directive, using Ulster antigen.

Total RNA extraction and viral RNA detection

RNA was extracted from tissue samples and allantoic liquid using a nucleic acid extraction workstation BioSprint 96 (Qiagen, Hilden, Germany), according to the manufacturer’s instructions. The presence of Newcastle disease virus was tested by real-time RT-PCR using the method described by Fuller and colleagues [12], which targets the L-gene. The reaction was performed in a final volume of 25 µl with 500 ng of total RNA, 25 pmol of each primer and 10 pmol of probe, using QIAGEN OneStep RT-PCR Kit (Qiagen, Hilden, Germany) or AgPath-ID One-Step RT-PCR kit (ThermoFisher Scientific, Waltham, USA), according to the manufacturer’s protocol. The amplification program included reverse transcription for 30 min at 50°C (OneStep Qiagen) or 15 min at 45°C (AgPath-ID) and activation of Taq polymerase at 95°C for 15 min (OneStep Qiagen) or 10 min (AgPath-ID), followed by

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50 cycles of denaturation at 95°C for 10 s, annealing at 50°C for 30 s and extension at 72°C for 30 s.

The birds tested negative for avian influenza by RT-PCR [13].

F-gene fragment amplification and sequencing

A fragment of 561 bp encompassing the 5’end of F-gene was amplified by RT-PCR using primer F (5’-TTAGAAAAAACACGGGTAGAA-3’) designed for this study and primer 2, described by Oberdofer [14]. The reaction was performed with 500 ng of total RNA and 25 pmol of each primer, using AgPath-ID One-Step RT-PCR kit (ThermoFisher Scientific), according to the manufacturer’s protocol. The amplification program included reverse transcription for 15 min at 45°C and activation of Taq polymerase at 95°C for 10 min, followed by 50 cycles of denaturation at 95°C for 30 sec, annealing at 55°C for 30 sec, and extension at 72C for 45 sec. A final extension was performed at 72°C for 5 min, and the expected fragments were purified by agarose gel electrophoresis, excised and purified using NZYGelpure kit (NZYTech, Lisboa, Portugal).

DNA sequencing was performed using a BigDye Terminator cycle sequencing kit (Applied Biosystems, Foster City, CA, U.S.A) according to the manufacturer’s instructions, with primers F and 2 used in the amplification reaction. The nucleotide sequences of the amplicons were determined on an automated 3130 Genetic Analyzer system (Applied Biosystems) and assembled with SeqScape v2.5 software (Applied Biosystems).

Phylogenetic analysis

To determine the genotype of the Newcastle disease virus strains detected in the July 2015 outbreak in Porto Santo Island, the nucleotide sequences obtained were compared with 68 sequences from other NDV isolates, representatives of all genotypes, retrieved from GenBank. The evolutionary history was conducted in MEGA6 [14], by using the best-fit model also obtained in MEGA6. Maximum Likelihood method based on the Kimura 2-parameter model [15] was used. Initial trees for the heuristic search were obtained by applying the Neighbor-Joining method to a matrix of pair wise distances estimated using the Maximum Composite Likelihood (MCL) approach. A discrete Gamma distribution was used to model evolutionary rate differences among sites.

Once the genotype was known, was determined by the subgenotype of the strains from Porto Santo, another phylogenetic analysis were included representatives of eight subgenotypes within genotype VI (a-c and e-i). The analysis was performed as described above.

Accession numbers

GenBank accession numbers KX831612, KX831613 and KX831614 were respectively attributed to partial F-gene nucleotide sequences of strains 16919-15, 17991-15 and 19095-15 of the Newcastle disease viruses detected in Porto Santo Island, Portugal 2015.

RESULTSEpidemiological data

An epidemiological survey was initiated as soon as the

presence of NDV was confirmed in the carcasses of the three pigeons. Figure 1 shows the Porto Santo Island map with the totality of the poultry backyard farms in the island. The results from the survey are summarized in Table (1). NDV was also detected in two other birds found dead, one pigeon and one turtle dove. Overall, 172 samples from 14 different species were submitted to viral investigation disclosing a 2.9% viral prevalence.

On the other hand, NDV antibodies were detected in 14 out of 42 serum samples tested (30.4% seroprevalence), mainly from chicken (11 out of 38) and duck (3 out of 6). Beside these species, only one serum from turkey and another from a passerine were investigated, both testing negative for NDV antibodies. The hemagglutination inhibition titers obtained for the three positive duck sera (24) were lower than the titers found in the eleven chicken serum samples, which ranged from 24 to 29 with more samples showing titers of 26 (n=3) and 27 (n=3). Although the titers found in ducks are usually low, this apparent difference in the titers obtained for the two species may be biased by the sampling sizes (≈6x more chickens than ducks).

Anatomic and histopathological data

The three pigeons presented cachexia. At necropsy, one of the birds showed congestion of lungs and liver, while the other two did not evidence gross lesions.

Table 1: Total of positive and negative samples tested, with indication of the species and matrix used for real-time PCR. Hemagglutination titers of positive serum samples obtained in hemagglutination inhibition are indicated as footnote.

SpeciesRT-PCR IH

Matrix POS NEG POS NEG

Pigeon Viscera Swabs

4-

109 - -

Chicken Viscera Swabs

--

167 11a 27

Turtle dove Viscera Swabs

1-

12 - -

PasserinesViscera SwabsFeces

---

125

- 1

Psittacines SwabsFeces

--

411 - -

Duck Swabs - 34 3b 3

Turkey Swabs - 8 - 1

Goose Swab - 1 - -

Quail Swabs - 4 - -Mandarin

duck Swabs - 3 - -

Pheasant Swab - 1 - -

Rhea Swab - 1 - -

Falcon Viscera - 1 - -

Swan Feces - 1 - -

TOTAL 5 167 14 32aHemaglutination titers: 16 (n=1), 32 (n=2), 64 (n=3), 128 (n=3), 256 (n=1) and 512 (n=1)bHemaglutination titers: 16 (n=3)

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0/11/311/8/58 0/17/60

0/12/145

0/22/440/14/31

0/2/31

0/12/84

0/14/64

3/37/249

0/7/26

0/11/62

1/20/1

0/2/28

Farms with no NDV-postive birds detectedFarm with NDV-postive pigeonBotanic park - index case

Wild birds found dead, including the NDV-positive turtle doveWild NDV-negative bird found dead

A - Virological survey

1/3/310/2/58

2/8/60

2/4/145

1/7/441/3/31

0/1/31

2/2/84

1/7/64

2/3/249

2/5/26

0/1/62

1/20/1

0/0/28

Farms with birds showing anti-NDV antibodiesFarms without birds showing anti-NDV antibodies

Botanic park - index case

Wild birds found dead, including the NDV-positive turtle doveWild NDV-negative bird found dead

B - Serological survey

1 Km

1 Km

Figure 1 Porto Santo Island map with the totality of the poultry backyard farms represented by circles. The star points the location of the park where the first three pigeons were detected (index case). Black circle represents the farm of the second NDV-positive victimized pigeon (case 2). Triangles indicate the locations where wild birds were found dead; black triangle corresponds to the origin place of the NDV-positive wild turtle dove (case 3). The numbers next to the symbols refer to the positive cases/samples tested/total of birds in the farm. A-Virological survey; B-Serological survey.

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Lung congestion was confirmed by histopathology (Figure 2A). In the liver of one of the birds, the presence of focal and periportal mononuclear inflammatory cell infiltration was observed (Figure 2B).

Virus isolation

Virus isolation was achieved in embryonated SPF chicken eggs after three blind passages. Isolation was subsequently confirmed by rapid hemagglutination (HA) and hemagglutination inhibition (HAI) tests with polyclonal NDV anti-serum. Viral isolation in eggs were also confirmed by real-time RT-PCR and sequencing.

Nucleotide sequence analysis

The 561 bp-long nucleotide sequence of the three pigeon isolates obtained from a pool of viscera (16919-15), as well as other pigeon (17991-15) and one turtle dove (19095-15) sequences were determined and found absolutely identical, suggesting a single introduction on the island. In order to compare our sequences with a higher number of other isolates, a smaller genomic region comprising the first 525 nucleotides of the fusion protein encoding gene was selected. A Blast (Basic Local Alignment Search Tool) analysis was performed using the NCBI platform (http://blast.ncbi.nlm.nih.gov/Blast.cgi) with a maximum of 20000 target sequences. The percentage of similarity between the strains characterized in this study and the other strains publically available found worldwide, ranged between 82% and 99%, revealing the high variability in this genomic region. Strain HG424625 obtained from a pigeon in Nigeria 2013 was the most similar to the Portuguese isolates, with 520 out of 525 nucleotides identical (99% similarity). Based on this smaller region, the isolates from Porto Santo share 97% similarity (511 out of 525 nucleotides) with three strains obtained from dove in Italy, in 2010 and 2011 (JN638234, JN638235 and JN638236). The percentage similarity with the remainder strains was lower than 91%. This indicates that the known strains from Portugal and the strains from Nigeria and Italy referred above are fairly different from the other known strains identified worldwide. Comparing with other strains from Portugal, also high variability was observed, except with two strains detected in 2016 in Portugal mainland (99% and 97% similarities).

The blast analysis revealed high variability all over the 525 nt-long fragments, including in the cleavage site encoding region. No singleton mutations are observed in the sequence of the three isolates from Porto Santo. The amino acid similarity between the three isolates from Porto Santo and the other strains ranged between 73% and 99%. As expected, the higher similarity was obtained with strains from Nigeria (HG424625) and Italy (JN638234, JN638235 and JN638236), reinforcing the close proximity of these viruses.

As regards to the cleavage site, the amino acid sequence deduced for the 2015 outbreak isolates is 112RRQKRFVGA120. This sequence fits within the consensus sequence of the F protein cleavage site of both velogenic and mesogenic strains, namely 112(R/K)RQ(R/K)RF117 [8]. The intracerebral pathogenicity index (ICPI) would have to be determined to unravel which of these two types caused the outbreak.

Phylogenetic analysis

Newcastle disease viruses can be phylogenetically divided into two distinct classes (class I and class II). Class I, composed of a single genotype encompasses only avirulent NDV strains (except APMV-1/chicken/Ireland48/904) and is found mainly in waterfowl.

Class II NDV are currently divided in 18 genotypes [7].

To investigate the origin of the Newcastle disease viruses detected in Porto Santo in 2015, a Maximum Likelihood phylogenetic analysis was performed using the three sequences obtained in this study and representatives of the eighteen genotypes previously referred in a total of 71 NDV genomic sequences. Class I NDV08-004 was included as an outer group.

In the consensus tree (Figure 3), the eighteen known clusters are clearly identified. The isolates from Porto Santo grouped within cluster VI, together with strains from Asia, Europe, Africa and America, obtained in chicken, dove and pigeon between 1972 and 2014. The inclusion of the strains isolated in Porto Santo in this genotype VI is in accordance with Susta et al. [16], who reported in 2015 that the genotypes involved in the most recent outbreaks detected in Europe belong to genotype VI. Genotypes V and VII are also involved in recent outbreaks, but in other areas of

A B

Figure 2 Histopathology of pigeon tissues (H&E, x200 magnification). A - Lung showing extensive congestion of the parenchyma and blood vessels (black arrows point to congested pulmonary arteries). B - Liver with periportal mononuclear inflammatory cell infiltration.

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X

XVI

XVIII

XVIIXIII

XII

II

IX

III

XIIV

VIII

V

VII

VI

KX831612-Pigeon/PRT/2015-16919-15KX831613-Pigeon/PRT/2015-17991-15KX831614-Turtledove/PRT/2015-19095-15

HG424625-Pigeon/NGA/2013-NIE13-005JN638235-NDV/DOVE/IT/11RS100_104VIR/2011JN638234-Dove/ITA/2011-11RS98_102VIRJN638236-Dove/ITA/10RS6171_7154VIR/2010KJ865707-Dove/ITA/10RS6171_7154VIR/2010

FJ480825-Pigeon/CHN/2005-JS/1AY288996-Pigeon/ITA/2000-1166JX901110-Pigeon/BEL/1998-98-248

KJ808819-Pigeon/CHN/2013-BJ2013KT381606-Pigeon/CHN/2014-GZ289

AB465606-Chicken/JPN/1985-Ibaraki/85AF458021-Chicken/CHN/1994-JX-1/94

AY562988-Chicken/USA/1972-1083(Fontana)AF358786-Chicken/TWN/2000-TW/2000AF234031-Unknown/TWN/1999-TW/99-156

AB853927-Chicken/JPN/1999-Ibaraki/SG106DQ682445-Goose/CHN/2004-SD/5/04/GoDQ682437-Goose/CHN/2003-JS/1/03/Go

AY028995-Unknown/CHN/1996-A7/96AF458014-Chicken/CHN/1999-JX-2/99

JN800306-Poultry/PER/2008-1918-03JN627508-Goose/CHN/2011-GD450

AY865652-Little tern/RUS/2001-2755GU182331-Chicken/PAK/2007-33FJ772475-Chicken/NER/2008-2602-605JX546243-Chicken/BEM/2009-373GC

JX390609-Chicken/TGO/2009-AKO18FJ772455-Avian/MRT/2006-1532-14JF966388-Chicken/MLI/2008-ML225 08

HM117720-Chicken/MEX/2005-P05-Mexico2005AY288999-Chicken/MEX/1996-37821

AY562990-Mixed species/USA/1971-Largo/71AY288989-Unknown/USA/1993-anhinga/USA(FL)/44083

GQ288388-Cormorant/USA/1992-92-23071/1997FJ751919-Chicken/CHN/1985-QH4FJ751918-Chicken/CHN/1979-QH1

JX119193-Chicken/DOM/2008-499-31JX915242-Chicken/DOM/1986-28138-4

M24702-Chicken/GBR/1933-Herts/33 (propagated in eggs)AY741404-Chicken/GBR/1933-Herts/33EU293914-Unknown/CHN/1944-Italien

HQ266602-Chicken/MDG/2008-MG_725_08HQ266603-Chicken/MDG/1992-MG_1992

M18456-Chicken/JPN/1951-MiyaderaFJ430159-Chicken/CHN/2005-JS/7/05/Ch

FJ430159-Chicken/CHN/2005-JS/7/05/ChEF201805-Unknown/CHN/Unknown-MukteswarFJ430160-Goose/CHN/2005-JS/9/05/GoFJ436302-Chicken/CHN/1946-F48E8FJ436305-Chicken/CHN/1997-JS/1/97/ChFJ436304-Chicken/CHN/1985-FJ/1/85/ChFJ436303-Chicken/CHN/1986-ZJ/1/86/ChKT381605-Duck/CHN/2014-YF18

HM125898-Duck/CHN/2004-WDK/JX/7793/2004AY562991-Chicken/GBR/1967-Ulster

GQ918280-Gull/SWE/1994-BHG/Sweden/94AY935499-Unknown/AUS/2005-I-2

M24693-Chicken/AUS/1966-Que-1966AF077761-Unknown/USA/1946-LaSotaKT381600-Duck/CHN/2014-YF24EU289028-Turkey/USA/1987-VG/GAFJ939313-Chicken/EGY/2005-1/2005GU978777-Chicken/USA/1978-U.S.(TX)/GB/1948

M24697-Unknown/GBR/Unknown-BEA-45X04719-Chicken/Unknown/Unknown-Beaudette C

GQ288391-Mottled duck/USA/2001-01-130FJ705464-Mallard/USA/2004-04-411

FJ794269-NDV08-004 China

92

95

92

98

100

100

9999

68

95

100

99

100

100

99

97

98

99

100

96

65

100

88

70

97

98

8256

74

94

99

54

98

9090

67

10

56

65

54

54

62

60

0,1

I

XV

XIV

Figure 3 Molecular phylogenetic analysis of a 525 bp fragment of the F-gene encoding sequence of Newcastle disease viruses representatives of the eighteen genotypes.The evolutionary history was inferred by using the Maximum Likelihood method based on the Kimura 2-parameter model [23]. The tree with the highest log likelihood (-6312,3893) is shown. The percentage of trees in which the associated taxa clustered together is shown next to the branches. Initial tree(s) for the heuristic search were obtained by applying the Neighbor-Joining method to a matrix of pair wise distances estimated using the Maximum Composite Likelihood (MCL) approach. A discrete Gamma distribution was used to model evolutionary rate differences among sites (5 categories (+G, parameter = 0,4432)). The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. The analysis involved 71 nucleotide sequences. Evolutionary analyses were conducted in MEGA6 [15]. Class I strain FJ794269 was included as an outer group. Bootstrap values higher than 50 are indicated next to the branches.

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VIi

VIc

VIg

VIh

VIa

VIb

VIf

VIeFJ480825-Pigeon/CHN/2005-PG/CH/JS/1/05

JQ290284-Pigeon/CHN/2009-P/Anhui/1/09AF358785-Pigeon/CHN/1998-Ch/98-1

JN942022-Chicken/USA/2004-309968/2004JN872182-Pigeon/USA/1998-12339/1998

JN872186-Pigeon/USA/1991-Indiana/18002/1991AY471785-Kestrel/ARE/1999-PAEKE99364

JF749831-Pigeon/URY/2008-Montevideo/02/08FM200798-Parrot/NGA/2007-NIE139/2007

AY445669-Unknown/ZAF/2002-ZA469/PPMV1/02EF030957-Dove/ZAF/2006-DOZA06N549

JN967788-Pigeon/USA/1985-Maryland/11936/1985AJ880277-Pigeon/ITA/1982

AY734535-Pigeon/ARG/1999-Tigre_6/99JN638234-Dove/ITA/2011-11RS98_102VIRJN638236-Dove/ITA/2010-10RS6171_7154VIRJN638235-Dove/ITA/2011-11RS100_104VIR

HG424625-Pigeon/NGA/2013-NIE13-005KX831614-Turtledove/PRT/2015-19095-15KX831612-Pigeon/PRT/2015-16919-15KX831613-Pigeon/PRT/2015-17991-15

JF824013-Pigeon/RUS/2009-0267/09JQ039385/Dove/NGA/2007-VRD07-163/2007

JQ039389-Pigeon/NGA/2007-VRD07-369/2007GQ507801-Chicken/KOR/1989-Kr-102/89

AB465606-Chicken/JPN/1985-Ibaraki/85HQ839733-Chicken/SWE/1995-Chicken_Sweden_95

98

98

97

100

9399

97

100

92

70

99

91

96

87

94

79

0.02

VIc

Figure 4 Molecular phylogenetic analysis of a 354 bp fragment of the F-gene encoding sequence of Newcastle disease viruses representatives of eight sub-genotypes of genotype VI.The evolutionary history was inferred by using the Maximum Likelihood method based on the Kimura 2-parameter model [23]. The tree with the highest log likelihood (-1684,5532) is shown. The percentage of trees in which the associated taxa clustered together is shown next to the branches. Initial tree(s) for the heuristic search were obtained by applying the Neighbor-Joining method to a matrix of pairwise distances estimated using the Maximum Composite Likelihood (MCL) approach. A discrete Gamma distribution was used to model evolutionary rate differences among sites (5 categories (+G, parameter = 0,5992)). The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. Evolutionary analyses were conducted in MEGA6 [15]. Bootstrap values higher than 50 are indicated next to the branches.

the world (V - Central and South America; VII – China and South Africa) [16]. Genotype VI strains were identified for the first time in 1960 and until 1985 were the most prevalent in poultry flocks in Asia [17]. From mid-1980s to late 2000 genotype VI was also predominant, but in pigeons and wild birds [18]. It is known that the prevailing PPMV-1 strains belong to genotype VI [19]. All over the years, this genotype was responsible by repeated epizootic episodes separated by short temporal intervals [20].

Genotype VI can be divided into nine subgenotypes (a-i) [19]. To determine in which subgenotype the strains of this study are included, a second Maximum Likelihood tree, also based in F gene was constructed with representatives of eight subgenotypes (VIa-VIc and VIe-VIi) (Figure 4). In order to include a maximum number of sequences, the analysis was limited to a region encompassing 354 nucleotides. As expected, the isolates from Porto Santo grouped within subgenotype VIi, together with the strains from Nigeria (HG424625) and Italy (JN638234-6) with which they shared the highest nucleotide similarity.

DISCUSSION AND CONCLUSIONThe detection of a virulent strain of Newcastle disease virus

in three wild pigeons in Porto Santo Island led the Portuguese veterinarian authorities to declare an outbreak (Animal Disease Notification System, ADNS-2015-07-20) and to implement an

epidemiological survey all over the island. These first detections occurred following abnormal mortality had been registered in turtle dove (n=11), but no conclusions could be taken regarding the role of this last species the 2015 outbreak, since advanced putrefaction hampered laboratorial diagnosis. However, turtle dove is not a resident species in the Island. It winters in southern Africa [21], namely in southern Mauritania, western Mali and the inner Niger delta [22] being observed in Portugal, including Porto Santo, between April and September. A high nucleotide similarity (99%) was observed between the Porto Santo 2015 strains and a strain from Nigeria (HG424625). Since Nigeria is used as wintering destination for turtle dove, the chance that the outbreak may relate with migration from Africa, should be considered. It is estimated that turtle dove takes about 15 days to travel from its winter habitat in Africa to Portugal [21]. Since the incubation period of ND may vary between two and 15 days [5], it is possible that the turtle doves flew infected with the Nigerian strain from Africa and subsequently contaminated local birds, leading to the dissemination of the virus in Porto Santo. Furthermore, Morocco and Algeria are used as spring stopover sites by this migratory bird [21], but since no significant nucleotide similarity (84%) was observed with the currently available sequence from Morocco (EU604259), no epidemiological association could be established.

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Besides turtle dove (Streptopelia turtur), other nonresident bird species such as mallard (Anas platyrynchos), Scopoli’s shearwater (Calonectris diomedea), Harcourt’s storm petrel (Oceanodroma castro), Bulwer’s petrel (Bulweria bulwerii) and common tern (Sterna hirundo), visit Porto Santo in summer. All these species are susceptible to Newcastle disease virus infection and can be reservoirs for the virus.

The traditional poultry farming in Porto Santo is mainly backyard poultry, with no industrial holdings found in the island. The contact with land-based wild birds is favoring viral transmission. Furthermore, free-living migratory waterfowls may carry virulent NDV strains [19], therefore constituting also a risk for NDV introduction. However, circulation of the virus in poultry is essential so that some NDV strains may exhibit their potential pathogenicity assessed by the cleavage site sequence. The low density of domestic birds in the island may have hampered the dissemination of the disease.

The detection of NDV-antibodies by HAI in chicken serum samples from different breeders demonstrated the virus circulation in poultry all over the island. A reduced number of birds tested positive by virus isolation and RT-PCR, with low viral charges (mean Cq 32). It is likely that these low viral charges due to limited viral multiplication allowed an effective antibody response that abrogated the ongoing dissemination of the virus in the island.

Although the birds affected by this outbreak were likely wild pigeons, prophylaxis of ND in pigeons through inactivated vaccines can be successful in PPMV-1 control, if accompanied by effective biosecurity actions and proper diagnostic of ND [10]. Despite vaccination was not practiced in Porto Santo Island before the outbreak, fast screening of the entire island poultry and wild birds along with evidences that effective humoral response developed after contact with the virus led to the efficient control of the outbreak.

More than one year after the last, and so far unique, NDV detection in Porto Santo Island, and following vaccination was implemented by the veterinarian authorities, no more mortality was registered.

ACKNOWLEDGMENTSThis work was supported by the Portuguese Fundação para a

Ciência e a Tecnologia under Grant SFRH/BPD/108281/2015 to A.M. Henriques. The authors are thankful to Arminda Batista, Ana Santos, Fátima Cordeiro, Ricardino Ferreira, Maria João Teixeira, Rosário Ferreira and Helena Carvalho for excellent technical support.

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Henriques AM, Fagulha T, Barros SC, Ramos F, Duarte M, et al. (2017) Newcastle Disease Virus 2015 Outbreak in Porto Santo Island, Portugal. Ann Virol Res 3(1): 1025.

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